Intestinal pathology caused by Shiga toxin (Stx)-producing enterohemorrhagic E.coli (EHEC), important food- borne pathogens, causes ~ 35% of all bloody diarrhea in the USA and life-threatening systemic complications, including the hemolytic uremic syndrome (HUS). EHEC is a particularly worrisome pathogen, because the numbers of outbreaks continue to increase and there is no effective specific therapy for this illness which is lethal in up to 10% of children who develop HUS. Thus, there is a great need to better understand the pathogenesis of this infection and to develop new therapeutic approaches to treat Stx-induced intestinal and systemic complications. The EHEC-induced diseases required that Stx must be endocytosed by the enterocytes, cross the intestinal epithelial barrier and enter the bloodstream. We have postulated that an antibiotic-independent method to prevent consequences of EHEC infections might be based on inhibiting Stx1 uptake and transcytosis across the intestinal epithelium. We have showed that the glycosphingolipid Gb3, the only known Stx1 receptor is not expressed in normal human intestinal epithelial cells. This recognition has required a rethinking of the pathogenesis of the toxin in EHEC diseases and provides the basis for the proposed studies. In this application using intestinal tissue from EHEC-infected humans, a well established model of rabbit cecal EHEC infection, and a cell culture model we will study the mechanisms of Stx1 uptake by intestinal epithelial cells, toxin transcytosis through the epithelium and the direct damage of epithelial cells by toxin. In Aim 1 we will determine the amount and distribution of Stx1 and Stx2 in intestinal tissue from patients with EHEC infection to establish a correlation between the outcome of disease (intestinal vs. systemic) and the toxin pattern in tissue. In Aim 2 we will characterize the molecular mechanism of Stx1 and Stx2 uptake by Gb3 receptor negative intestinal epithelial cells and toxin transcytosis. The role of Src and actin-binding proteins involved in macropinocytosis will be explored. Aim 3 will determine the Stx1 trafficking pathway that leads to apoptosis of intestinal epithelial cells and the role of secreted galectin-3 in apoptosis. The results gained from these experiments will not only help to elucidate the molecular mechanisms of Stx1 interaction with receptor free intestinal epithelial cells but will also contribute to our understanding of mechanisms of interaction between broad varieties of bacterial products. PUBLIC HEALTH RELEVANCE: Shiga toxin (Stx) producing E. coli (EHEC)-related illnesses, including hemolytic uremic syndrome (HUS), are of growing medical concern in the USA. To cause HUS, Stx must spread systemically. In this grant, we will characterize the mechanisms of Stx uptake by the intestinal epithelium and develop pharmacologic methods to stop this uptake, which should allow development of therapy against EHEC.